Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon-subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [C-14]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha-subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha-subunits) and with the delta-subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta-subunit to both alpha and beta-subunits. Reaction of the mutants with TFPAM's also cross-linked-delta to alpha and beta-and in addition formed covalent links between Cys10 of the epsilon-subunit and the gamma-subunit and between CYS108 of the epsilon-subunit and the alpha-subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta-cross-linked products. In the presence of ATP + EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%). In the presence of ATP + Mg2+ the yield of epsilon-Cys10-gamma was lower than in ATP + EDTA (only 22%) and the yield of cross-linking between epsilon-Cys108 and alpha was higher (now around 30%). These changes in cross-linking of epsilon to near-neighbor subunits support previous work [Mendel-Hartvig, J., & Capaldi, R. A. (1991) Biochemistry 30, 1278-1284] in showing that there are ligand-dependent conformational changes and/or binding changes of the epsilon-subunit. Cross-linking of the epsilon-subunit to gamma had very little effect on ATPase activity, while cross-linking of the epsilon-subunit to an alpha-subunit inhibited ATPase activity dramatically.
